Identification of proteins that interact with PKA Regulatory subunit from Saccharomyces cerevisiae

FIORELLA GALELLO; SILVIA MORENO; SILVIA ROSSI

Los Cocos, Córdoba

Simposio; First South American Spring Symposium in Signal Transduction and Molecular Medicine (SISTAM),; 2010

SISTAM

Subcellular targeting through the association with adaptor and scaffolding proteins has emerged as a key mechanism by which cells maintain signaling specificity. Compartmentalization of cAMP-PKA pathway is maintained by the clustering of cAMP signaling enzymes in discrete units by the A kinase anchoring proteins (AKAPs) through their interaction with regulatory subunit (R). No anchoring proteins had been characterized up to now in S. cerevisiae. We have identified proteins that tether regulatory subunit (Bcy1) from yeast PKA using TAP-affinity purification and MALDI-TOF identification. The interactions were assessed by pull-down and peptide arrays. Although a-helices were predicted in all domains involved in the interaction with Bcy1, they do not present a perfect hydrophobic face. Similar residues to those that shape the binding pockets in mammalian AKAPs are present. The importance of these residues was assayed using peptide arrays. Positive charged residues present in the domain are determinant for the interaction. We demonstrate using BCY1 N-terminus mutants that the region 1-138 is necessary for the interaction with these proteins. The interaction in vivo was corroborated by pull-down and inmunoprecipitation assays